When an immunological solid phase method is used to measure or assay a certain antigen, a solid phase can be coated with antibodies against this antigen. This often occurs by physical adsorption of the antibodies on the inside of plastic tubes or wells in plastic plates ("coat" in the technical literature). When the antigen-containing test solution is placed in the tubes or wells, the antigen are selectively bonded (antibody-antigen reaction) to the adsorbed antibody. The tube or well can then be emptied and thoroughly washed. In this way antibody present on the surface can be transferred from the matrix (medium) in which it has been present, to a much more easily handled solid phase, where detection and quantification can be performed. With the well-known ELISA method, this is carried out with an additional antibody directed against the antigen. This antibody can be labelled with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase. The enzyme-labelled (enzyme-conjugated) antibody is placed in the tube or well and is bonded to the antigen present on the walls or surfaces. The excess enzyme-labelled antibody is washed away, and the remaining enzyme-labelled antibody is detected with an enzyme substrate that is converted to a colored product.
The variations of the above-mentioned ELISA technique are numerous, but the fundamentals are the same. In theory only the antigen is removed from its original matrix to a surface where it is uniformly exposed and is accessible to analysis.
In practice, there are always other components in the test solution that are bonded to this surface, either to the absorbed antibody or directly to the solid phase by some other mechanism. These non-specifically bonded components represent a significant source of error in the method, since they can interfere with the analysis to be performed and give rise to falsely positive or falsely high measurement results. In the traditional ELISA technique, non-antigen components in the sample which are bonded to the solid phase can absorb enzyme-conjugated antibody and give a falsely high measurement result. These non-antigen-mediated measurement results are believed to result from nonspecific absorption.
In qualitative analysis (e.g., testing only for the presence or absence of antibodies or antigens in a sample) elevated assay responses due to nonspecific effects will cause a false positive result. Attempts to reduce the false positive effects by elevating the discrimination level of the assay will cause increases in the numbers of false negative results. Thus, nonspecific effects in these assays create uncertainty in the diagnosis.
Previous techniques have attempted to reduce nonspecific absorption (or bonding) to a minimum. For example, the surface of the solid phase has been thoroughly "blocked" to make it incapable of absorbing additional components. In addition, the trapped antibodies have been enzymatically split and the antigen-bonding fraction ((Fab).sub.2- fragment) has been isolated and immobilized on the solid phase. However, total success has not been achieved with these techniques. Moreover, it should be noted that difficulties in using the ELISA technique have recently been noted to an increasing extent; see, for example, Gaffney et al., 1984, Thromb. Haemostas, 52 (1):96-97, Boscato, et al., 1986, Clin. Chem., 32 (8): 1491-1495